1. Field of the Invention
The present invention relates to flavivirus E proteins and their use in vaccines against flavivirus infection.
2. Background Information
Endemic dengue caused by one or more of the four types of dengue viruses is a major public health problem in many tropical and subtropical areas. In addition, sporadic dengue epidemics at times involving over a million individuals, continue to occur in these regions. Many other members of the flavivirus family are also etiologic agents of severe diseases such as yellow fever, Japanese encephalitis, St. Louis encephalitis, and tick-borne encephalitis.
Epidemic outbreaks, for example, caused by Japanese encephalitis virus (JEV) continue to pose serious public health problems in the densely populated regions of tropical and subtropical Asia. Transmitted by species of the Culex genus mosquitos, the disease is clinically manifested as encephalitis, often severe and with a high mortality rate among young children and elderly people. Further, permanent neurological sequelae of different severity can occur in a high percentage of patients who survive. JEV also infects domestic animals such as swine and horses. During the last two decades, immunization using an inactivated JEV vaccine has brought the disease under control in Japan, Korea and Taiwan. However, because of the high cost of manufacturing the vaccine, it is not readily available to those countries where it is needed the most.
Flaviviruses, including the dengue virus and the JEV, contain only three structural proteins, that is, a capsid protein (C, mol. wt. 12-14 kd) which binds to the positive strand genomic RNA forming the nucleocapsid, and two membrane associated proteins termed the small membrane protein (M, mol. wt. 7-8 kd) and the large membrane protein also called envelope glycoprotein (E, mol. wt. 55-60 kd) (Stollar, V. 1969. Studies on the nature of dengue viruses. IV The structural proteins of type 2 dengue virus. Virology 39:426-438). The envelope glycoprotein is the major virion antigen responsible for virus neutralization by specific antibodies and for several important antigenic properties such as binding to flavivirus-, dengue complex-, and type-specific antibodies (Clarke, D. H. 1960. Antigenic analysis of certain group B arthropod-borne viruses by antibody absorption. J. Exp., Med. 111:21-32. Roehrig, J. T., J. H. Mathew, and D. W. Trent. 1983 Identification of epitopes on the E glycoprotein of St. Louis encephalitis virus using monoclonal antibodies. Virology 128:118-126). Dengue and other flavivirus E's also exhibit a hemagglutinating activity that is presumably associated with virus attachment to the cell surface and subsequent virus uncoating (Sweet, B. H., and A. B. Sabin. 1954. Properties and antigenic relationships of hemagglutinins associated with dengue viruses. J. Immunol. 73;363-373). The full-length dengue type 4 virus E sequence contains 494 amino acids including two hydrophobic regions at the C-terminus, 15 amino acids and 24 amino acids in length, separated by an arginine. These hydrophobic sequences may serve to interact with the lipid membrane during virus assembly. Evidence from limited protease digestion of tick-borne encephalitis virus E glycoprotein suggest that the hydrophobic C-terminus is inserted into the lipid membrane exposing the bulk of the N-terminus of E on the virion surface.
The full-length E of dengue type 4 virus contains 12 cysteine residues, all of which are conserved in at least 20 flavivirus E's that have been sequenced. The most C-terminal cysteine in dengue type 4 E is at position 333 of the E sequence. Thus, 67% or more of the N-terminal E should contain all 12 cysteine residues. In flavivirus West Nile E, each of the 12 cysteines appeared to be involved in di-sulfide bond formation (Nowak, T., and G. Wengler 1987. Analysis of disulfides present in the membrane proteins of the West Nile flavivirus. Virology 156:127-137). In dengue type 4 E, two potential N-linked glycosylation sites are located at positions 67 and 153 and a third N-glycosylation site is present at position 471 within the C-terminal hydrophobic region that is probably not used. 31% N-terminal E contains both potential glycosylation sites. However, the E glycoproteins of 2 flaviviruses, West Nile virus and Kunjin virus, lack glycosylation sites suggesting that N-glycosylation is not essential to the antigenic, structural, and functional integrity of the flavivirus envelope glycoprotein (Coia, G., M. D. Parker, G. Speight, M. E. Byrne, and E. G. Westaway. 1988. Nucleotide and complete amino acid sequences of Kunjin virus: definitive gene order and characteristics of the virus-specified proteins. J. Gen. Virol. 69:1-21; and Wengler, G., E. Castle, U. Leidner, T. Nowak, and G. Wengler. 1985. Sequence analysis of the membrane protein v3 of the flavivirus West Nile virus and of its gene. Virology 147:264-274).
Results of epitope mapping with a library of monoclonal antibodies indicate that the antigenic structure of dengue E is similar to that of other flavivirus E's that contain several distinct antigenic sites as defined by serological specificity, functional activity, and competitive binding assay (Henchal, E. A., J. M. McCown, D. S. Burke, M. C. Sequin, and W. E. Brandt. 1985. Epitopic analysis of antigenic determinants on the surface of dengue-2 virus using monoclonal antibodies. Am. J. Trop. Med. Hyg. 34:162-169; and Heinz, F. X. 1986. Epitope mapping of flavivirus glycoproteins. Advance in Virus Research Vol. 31, pp. 103-168, K. Muramorosch, F. A. Murphy, and A. J. Shatkin (ed.)). All E's appears to be similar consisting of at least three nonoverlapping antibody-binding domains (antigenic sites). A majority of these sites are dependent on the protein conformation since fragmentation of E by protease digestion or chemical disruption of disulfide bonds abolishes antibody binding (Mandl, C. W., F. Guirakhoo, H. Holzmann, F. X. Heinz, and C. Kunz. 1989. Antigenic structure of the flavivirus envelope protein E at the molecular level, using tick-borne encephalitis virus as a model. J. Virol. 63:564-571; and Winkler, G., F. X. Heinz, and C. Kunz. 1987. Characterization of a disulfide bridge-stabilized antigenic domain of tick-borne encephalitis virus structural glycoprotein. J. Gen. Virol. 68:2239-2244). Moreover, the identification of two or more sites involved in neutralization, hemagglutination inhibition or passive protection indicates that these functions are not localized to a single domain on the E glycoprotein.
More recently, complete or nearly complete sequences of the genome of several dengue viruses as well as other major flaviviruses have been determined and their polyprotein sequences deduced (Castle, E., T. Nowak, U. Leidner, G. Wengler, And G. Wengler. 1985. Sequence analysis of the viral core protein and the membrane-associated proteins V1 and NV2 of the flavivirus West Nile virus and of the genome sequence for these proteins. Virology 145:227-236; Coia, G., M. D. Parker, G. Speight, M. E. Byrne, M. and E. G. Westaway. 1988. Nucleotide and complete amino acid sequences of Kunjin virus: definitive gene order and characteristics of the virus-specified proteins. J. Gen. Virol. 69:1-21; Deubel, V., R. Kinney, and D. W. Trent. 1986. Nucleotide sequence and deduced amino acid sequence of the structural proteins of dengue type 2 virus, Jamaica genotype. Virology 155:365-377; Gruenerg, A., W. S. Woo, A. Biedrzyca, and P. J. Wright. 1988. Partial nucleotide sequence and deduced amino acid sequence of the structural proteins of dengue virus type 2, New Guinea C and PUO-218 strains. J. Gen. Virol. 69:1391-1398; Hahn, Y. S., R. Galler, T. Hunkapiller, J. M. Dalyryple, J. H. Strauss, and E. G. Strauss. 1988. Nucleotide sequence of dengue 2 RNA and comparison of the encoded proteins with those of other flaviviruses. Virology. 162:167-180; Irie, K., P. M. Mohan, Y. Sasaguri, R. Putnak, and R. Padmanabhan. 1989. Sequence analysis of cloned dengue type 2 genome (New Guinea-C strain). Gene 75:197-211; Mackow, E., Y. Makino, B. Zhao, Y. -M. Zhang, L. Markoff, A. Buckler-White, M. Guiler, R. M. Chanock, and C. -J. Lai. 1987. The nucleotide sequence of dengue type 4 virus: analysis of genes coding for nonstructural proteins. Virology 159:217-228; Mason, P. W., P. C. McAda, T. L. Mason, and M. J. Founier. 1987. Sequence of the dengue-1 virus genome in the region encoding the three structural proteins and the major nonstructural protein NS1. Virology 161:262-267; Osatomi, K., I. Fuke, D. Tsuru, T. Shiba, Y. Sakaki, H. Sumiyoshi. 1988. Nucleotide sequence of dengue type 3 virus genomic RNA encoding viral structural proteins. Virus Genes 2:99-108; Pletnev, A. G., V. Yamshchikov, and V. M. Blinov. 1990. Nucleotide sequence of the genome and complete amino acid sequence of the polyprotein of tick-borne encephalitis virus. Virology 174:250-263; Rice, C. M., E. M. Lenches, S. R. Eddy, S. J. Shin, R. L. Sheets, and J. H. Strauss. 1985. Nucleotide sequence of yellow fever virus: implications for flavivirus gene expression and evaluation. Science 229:726-733; Sumiyoshi, H., C. Mori, I. Fuke, K. Morita, S. Kuhara, J. Kondou, Y. Kikuchi, H. Nagamutu, and A. Igarashi. 1987. Complete nucleotide sequence of the Japanese encephalitis virus genome RNA. Virology 161:497-510; Wengler, G., E. Castle, U. Leidner, T. Nowak, and G. Wengler. 1985. Sequence analysis of the membrane protein v3 of the flavivirus West Nile virus and of its gene. Virology 147:264-274; and Zhao, B., E. Mackow, A. Buckler-White, L. Markoff, R. M. Chanock, C. -J. Lai, and Y. Makino. 1986. Cloning full-length dengue type 4 virus DNA Sequences: analysis of genes coding for structural proteins. Virology 155;77-88). Comparison of amino acid sequences showed that there is significant sequence homology among the E glycoproteins of different flaviviruses. For example, between dengue type 4 virus and JEV, the amino acid homology is 44% whereas the amino acid homology varies from 60-74% among degne viruses. As expected, sequence homology of E was observed to be greatest among the different dengue serotypes.
Despite four decades of research effort, a safe and effective vaccine against flaviviruses such as dengue is still not available. However, several new strategies for vaccine development based upon the use of cloned dengue cDNA for synthesis of dengue protective antigens have yielded encouraging results. During recent studies, expression of cloned DNA that codes for the dengue type 4 virus structural proteins (C, M, and E) together with nonstructural protein NS1 was achieved during infection of: (i) eucaryotic cells with a vaccinia virus-dengue recombinant or (ii) insect cells with a baculovirus-dengue recombinant (Zhao, B., G. Prince, R. Horswood, K. Eckels, P. Summer, R. M. Chanock, and C. -J. Lai. 1987. Expression of dengue virus structural proteins and nonstructural protein SN1 by a recombinant vaccinia virus. J. Virol 61:4019-4022; and Zhang, Y. M., E. P. Hayes, T. C. McCarty, D. R. Dubois, P. L. Summers, K. H. Eckels, R. M. Chanock, and C. -J. Lai. 1988. Immunization of mice with dengue structural proteins and nonstructural protein NS1 expressed by baculovirus recombinant induces resistance to dengue encephalitis. J. Virol. 62:3027-3031). The vaccinia virus recombinant system was also employed to separately express dengue E or NS1 glycoprotein (Bray, M., B. Zhao, L. Markoff, K. H. Eckels, R. M. Chanock, and C. -J., Lai. 1989. Mice immunized with recombinant vaccinia virus expressing dengue 4 virus structural proteins with or without nonstructural protein NS1 are protected against fatal dengue virus encephalitis. J. Virol. 63:2853-2856; and Falgout, B., R. M. Chanock, and C. -J. Lai. 1989. Proper processing of dengue virus nonstructural glycoprotein NS1 requires the N-terminal hydrophobic signal sequence and the downstream nonstructural protein NS2A. J. Virol. 63:1852-1860). Infection of mice with a vaccinia virus recombinant expressing full-length E and NS1 glycoproteins [v(C-M-E-NS1-NS2A)], 93% of the N-terminal E sequence [v(93% E)], or only NS1 [v(NS1-NS2A)] induced complete or almost complete resistance to fatal encephalitis resulting from intracerebral inoculation of dengue type 4 virus (Bray, M., B. Zhao, L. Markoff, K. H. Eckels, R. M. Chanock, and C. -J., Lai. 1989. Mice immunized with recombinant vaccinia virus expressing dengue 4 virus structural proteins with or without nonstructural protein NS1 are protected against fatal dengue virus encephalitis. J. Virol. 63:2853-2856; and Falgout, B., M. Bray, J. J. Schlesinger, and C. -J. Lai 1990. Immunization of mice with recombinant vaccinia virus expressing authentic dengue virus nonstructural protein NS1 protects against lethal dengue encephalitis. J. Virol. 64: 4356-4363, 1990). Immunization of mice with baculovirus recombinant expressed E and NS1 also induced a similar level of resistance (Zhang, Y. M., E. P. Hayes, T. C. McCarty, D. R. Dubois, P. L. Summers, K. H. Eckels, R. M. Chanock, and C. -J. Lai. 1988. Immunization of mice with dengue structural proteins and nonstructural protein NS1 expressed by baculovirus recombinant induces resistance to dengue encephalitis. J. Virol. 62:3027-3031). However, v(C-M-E-NS1-NS2A) or v(93% E) or the baculovirus recombinant-infected cell lysate containing expressed E, consistently failed to induce detectable antibodies to E or induced only a very low level of such antibodies in mice (Bray, M., B. Zhao, L. Markoff, K. H. Eckels, R. M. Chanock, and C. -J., Lai. 1989. Mice immunized with recombinant vaccinia virus expressing dengue 4 virus structural proteins with or without nonstructural protein NS1 are protected against fatal dengue virus encephalitis. J. Virol. 63:2853-2856; and Zhang, Y. M., E. P. Hayes, T. C. McCarty, D. R. Dubois, P. L. Summers, K. H. Eckels, R. M. Chanock, and C. -J. Lai. 1988. Immunization of mice with dengue structural proteins and nonstructural protein NS1 expressed by baculovirus recombinant induces resistance to dengue encephalitis. J. Virol. 62:3027-3031). A more recent study showed that monkeys immunized with baculovirus-expressed E also failed to develop antibodies to E as detected by radioimmunoprecipitation and presumably for this reason only partial resistance to intravenous dengue virus challenge was induced (Lai, C. -J., Y. -M. Zhang, R. Men, M. Bray, R. M. Chanock, D. R. Dubois, and K. H. Eckels. 1990. Immunization of monkeys with baculovirus recombinant-expressed dengue envelope and NS1 glycoproteins induces partial resistance to challenge with homotypic dengue virus, p. 119-124. In F. Brown, R. M. Chanock, H. S. Ginsberg, and R. Lerner (ed.), Vaccines 90: Modern approaches to new vaccines including prevention of AIDS. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).
There has also been a recent report of a live, attenuated dengue-2 candidate vaccine, which induced neutralizing antibodies in 10 human volunteers, who suffered no untoward effects of immunization (Immunization with a live attenuated dengue-2 virus candidate vaccine, 16681-PDK53: clinical, immunological and biological responses in adult volunteers. Bull. Who 65:189-195, 1987). However, this report suggests that the candidate vaccine is heat-labile, as the vaccine was stored at -80.degree. C. prior to use, and aliquots for virus titration were kept on ice following immunization.
Such a vaccine would probably require a continuous "cold chain" for use in tropical areas. There is no basis for estimating the cost per dose for a live, attenuated vaccine, but it may be several dollars, a possibly prohibitive amount for mass immunization in "third world" nations. A vaccinia recombinant vaccine producing dengue virus antigens should share the characteristics of the vaccinia virus vaccine proven during successful global smallpox eradication campaign. That is, the vaccine should be safe, heat stable and easily administered, and have a low cost per dose. Thus, the low immunogenicity of E constitutes a major obstacle to the development of an effective dengue vaccine produced by recombinant DNA technology.